Otitis media is one of the most common diseases seen in pediatric practice and the most common reason for antibiotic prescriptions in the U.S. The long-term objectives of our research team are to elucidate the contribution of infectious pathogens, and their complex interactions with other otitis risk factors in the pathogenesis of and recovery from acute otitis media (AOM), and to identify possible strategies for more effective prevention and/or treatment. Respiratory viruses play an important role in AOM pathogenesis; AOM often occurs as a consequence of viral upper respiratory tract infection (URI). One way to prevent AOM is to prevent URI or stop its progress to AOM; these require knowledge and understanding of the process of AOM development after URI and on bacteria and viruses involved. Recent use of polymerase chain reaction (PCR) assays to detect viruses has generated new questions on asymptomatic infection, viral persistence, and multiple viral infections during URI and its AOM complication. In this revision proposal, we will use a recently developed, cost-effective, high throughput, PCR-based system to identify and quantify a broad spectrum of respiratory viruses and sequence the viruses, to study early life viral infections, virl persistence and viral - bacterial interactions in AOM pathogenesis. Aim 1: Determine the causative viruses and the incidence of asymptomatic and symptomatic viral URI in infants during the first 6 months of life and the effect of viral load and co-presence of pathogenic bacteria on URI symptoms and AOM development. Aim 2: Characterize rhinovirus and RSV that are detected repeatedly in sequential samples, to differentiate new infection from prolonged presence of virus nucleic acids in the respiratory tract using a cost-effective sequence-based screening paradigm. The parent grant supports an ongoing study of 300 infants with or without a known genetic risk, enrolled from near birth and followed to the first AOM episode or between 6-12 mos. of age. Nasopharyngeal specimens are collected monthly (mos. 1-6) to study the acquisition of bacterial pathogens and during symptomatic URI episodes and AOM. We will utilize these existing specimens for detection and quantification of 13 most common respiratory viruses. We will sequence the viruses that are detected repeatedly to differentiate new infection from prolonged presence. This study will determine the novel incidence of asymptomatic URI in young infants and define the role of asymptomatic URI in AOM pathogenesis. Quantitative PCR data will address the relationship between viral load and symptom severity and help indicate the likely pathogen in case of multiple virus detection; results will also provide an immediate benefit on PCR result interpretation during respiratory infections in clinical setting. Information generated from the study will be important to public health as it will have clinical implication an will lay the ground work for future design and development of novel strategies such as specific viral vaccines, use of antivirals or antibiotics to prevent URI and/or AOM to reduce the public health burden of these highly prevalent diseases.